The PII signal transduction protein of E. coli plays a central role in nitrogen regulation by virtue of its interaction with 3 bifunctional enzymes: uridylyltransferase/uridylyl-removing enzyme (UTase/UR), adenylyltransferase (ATase), and Nitrogen Regulator II (NRII). The UTase/UR enzyme catalyzes the reversible uridylylation of PII to PII-UMP in response to intracellular signals of nitrogen status. The ATase enzyme reversibly modifies glutamine synthetase (GS) and thereby controls GS activity. NRII is a kinase/phosphatase that controls the phosphorylation state and hence the activity of the transcription factor Nitrogen Regulator I (NRI), which is a transcriptional activator when phosphorylated. The PII protein serves as a diffusible regulatory subunit of ATase and NRII. Unmodified PH stimulates the adenylylation of GS by ATase, and stimulates the phosphatase activity of NRII. PII-UMP stimulates the deadenylylation of GS-AMP by ATase, but has no effect on NRII. Thus, the metabolically regulated uridylylation and deuridylylation of PII, catalyzed by the UTase/UR, regulates the both activity and biosynthesis of GS. The ability of PII to interact with the UTase/UR and with NRII is controlled by the binding of the small molecule effectors 2-ketoglutarate and ATP to PII. The activity of the UTase/UR enzyme is regulated by glutamine. We propose to study the interaction of PII with its protein receptors and small molecule effectors using genetic and biochemical approaches. We describe a plan for the isolation and biochemical characterization of mutant forms of PII specifically defective in each of its interactions. We also propose experiments designed to identify the functional role of various structural features of PII, such as the identification of the contacts between PII and its receptors and small molecule effectors. Finally, we propose experiments designed to elucidate the mechanistic basis for the consequences of PII interaction with each of its receptors. The results of these studies will reveal how the interactions of PII with its receptors and small molecule effectors brings about the regulation of glutamine synthetase. These studies should also provide a paradigm for the mechanisms by which activities of bifunctional signal-transducing enzymes may be controlled by diffusible regulatory subunits.